Endothelial cell derived Nitric oxide (NO) is an important regulator of blood flow and pressure. However,at high levels NO can inhibit a number of cellular enzymes including endothelial NO synthase (eNOS).The molecular mechanisms underlying this inhibition remain elusive. Understanding how NO inhibits eNOS is important due to the extensive clinical use of NO to treat a variety of cardiovascular disorders.Inhaled NO is used to treat some children born with pulmonary hypertension while NO donors are used to treat angina. However, eNOS inhibition limits the efficacy of these therapies, producing rebound pulmonary hypertension or nitrate tolerance respectively. Thus, the principal objective of this proposal is to elucidate the mechanisms by which NO exposure results in an inhibition of eNOS. Recent studies from this laboratory indicate that peroxynitrite-mediated nitration of eNOS, secondary to increased superoxide generation from eNOS itself, plays a major role in the inhibitory mechanism. To further investigate this we will use an integrated molecular, biochemical, and physiological approach. Initially we will determine how exogenous NO uncouples eNOS causing it to generate superoxide. Studies in Aim 1 will investigate the link between NO-mediated zinc release, PKC activation, and eNOS phosphorylation at Thr-495. Aim2 will investigate the link between NO-mediated p21 ras activation and eNOS phosphorylation at Serl 177. In addition, the expression of eNOS mutant proteins that mimic or prevent these phosphorylation events will be used to investigate their role in uncoupling eNOS. Studies in Aim 3 will then identify the nitration sites on eNOS and elucidate the mechanism by which peroxynitrite reduces eNOS activity. Finally, Aim 4 will determine if decreasing superoxide generation can ameliorate the rebound pulmonary hypertension in vivo using our lamb model of inhaled NO therapy. The successful completion of these studies should yield important information regarding the control of redox-mediated signal transduction and NOS post-translational regulation. The information garnered should prove to be significant in a number of physiological processes. Clinically, these studies may be important in interventions directed at pathophysiological disorders in which redox signaling is involved.